Nimtz, M. | bioRxiv Science

bioRxiv2020

Clarithromycin Exerts an Antibiofilm Effect against Salmonella typhimurium rdar Biofilm Formation, and Transforms the Physiology towards an Apparent Oxygen-depleted Energy and Carbon Metabolism

Upon biofilm formation, production of extracellular matrix components and alteration in physiology and metabolism allows bacteria to build up multicellular communities which can facilitate nutrient acquisition during unfavorable conditions and provide protection towards various forms of environmental stresses to individual cells. Thus, bacterial cells become tolerant against antimicrobials and the immune system within biofilms. In the current study, we evaluated the antibiofilm activity of the macrolides clarithromycin and azithromycin. Clarithromycin showed antibiofilm activity against rdar (red, dry and rough) biofilm formation of the gastrointestinal pathogen Salmonella typhimurium ATCC14028 Nalr at 1.56 M subinhibitory concentration in standing culture and dissolved cell aggregates at 15 M in a microaerophilic environment suggesting that the oxygen level affects the activity of the drug. Treatment with clarithromycin significantly decreased transcription and production of the rdar biofilm activator CsgD, with biofilm genes such as csgB and adrA to be consistently downregulated. While fliA and other flagellar regulon genes were upregulated, apparent motility was downregulated. RNA sequencing showed a holistic cell response upon clarithromycin exposure, whereby not only genes involved in the biofilm-related regulatory pathways, but also genes that likely contribute to intrinsic antimicrobial resistance, and the heat shock stress response were differentially regulated. Most significantly, clarithromycin exposure shifts the cells towards an apparent oxygen- and energy-depleted status, whereby the metabolism that channels into oxidative phosphorylation is downregulated, and energy gain by degradation of propane 1,2-diol, ethanolamine and L-arginine catabolism is upregulated. This initial analysis will allow the subsequent identification of novel intrinsic antimicrobial resistance determinants.

microbiology↗

bioRxiv2020

Crystal structure of NirF: Insights into its role in heme d1 biosynthesis

Certain facultative anaerobes such as the opportunistic human pathogen Pseudomonas aeruginosa can respire on nitrate, a process generally known as denitrification. This enables denitrifying bacteria to survive in anoxic environments and contributes e.g. to the formation of biofilm, hence increasing difficulties in eradicating P. aeruginosa infections. A central step in denitrification is the reduction of nitrite to nitrous oxide by nitrite reductase NirS, an enzyme that requires the unique cofactor heme d1. While heme d1 biosynthesis is mostly understood, the role of the essential periplasmatic protein NirF in this pathway remains unclear. Here, we have determined crystal structures of NirF and its complex with dihydroheme d1, the last intermediate of heme d1 biosynthesis. We found that NirF forms a bottom-to-bottom {beta}-propeller homodimer and confirmed this by multi-angle light and small-angle X-ray scattering. The N-termini are immediately neighbored and project away from the core structure, which hints at simultaneous membrane anchoring via both N-termini. Further, the complex with dihydroheme d1 allowed us to probe the importance of specific residues in the vicinity of the ligand binding site, revealing residues not required for binding or stability of NirF but essential for denitrification in experiments with complemented mutants of a {Delta}nirF strain of P. aeruginosa. Together, these data implicate that NirF possesses a yet unknown enzymatic activity and is not simply a binding protein of heme d1 derivatives.

biochemistry↗